JP2014066729A - Diagnostic device and method of use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of performing diagnostic analysis on tissue samples, a microfluidic device, and a microfilter device.SOLUTION: The present invention relates to methods of diagnosing samples as well as various microfluidic, microcentrifuge and microfilter devices. In one embodiment, the present invention provides a method of diagnosing neurodegenerative diseases using mitochondrial and/or platelet samples. In another embodiment, the present invention provides a microfluidic device that selectively captures and analyzes a desired amount of target biological particles. Other embodiments include a microfluidic kit for the diagnosis of Alzheimer's Disease and/or mild cognitive impairment, comprising a microfluidic diagnostic device and instructions for use.

Description

  The present invention relates to methods for performing diagnostic analysis on tissue samples, and microfluidic and microfilter devices.

  All publications herein are incorporated into the reference to the same extent as if each individual publication and patent application were clearly and individually indicated to be incorporated into the reference. The following description includes information that may be useful in understanding the present invention. No admission is made that any information provided herein is prior art or related to the presently claimed invention, and any publication explicitly or implicitly mentioned is prior art. Is not allowed.

Originally developed to produce large scale integrated circuits, microfabrication technology has also stimulated research and development efforts to manipulate and analyze complex biological fluids at the nanoliter and microliter scales . The development of miniaturized diagnostic devices for clinical analysis of blood and other body fluids is an important application of this emerging technology. ^1-6 For example, point-of-use blood glucose monitoring systems have demonstrated the ability of miniaturized clinical chemistry techniques to improve patient care and healthcare delivery methods. ^{7, 8}
Many current diagnostic techniques require sample preparation prior to analysis, and microfluidic devices can be designed and fabricated with an integrated isolation process upstream of the analyte detection region . For example, glucose test strips isolate plasma from whole blood by glass fiber filters or microporous membranes. ^Nine researchers have reported many advances in lab-on-chip diagnostics, but only a few reports on microfluidic fractionation of samples. ¹⁰ For example, Brody et al. ¹¹ suggested using a microfabricated filter device to separate plasma from whole blood and reported filtration of a suspension of microspheres. Wilding et al. ¹² demonstrated using microfilters to measure leukocytes for genetic analysis, and Duffy et al. ¹³ proposed centrifugation on a rotating “lab disc”. Nevertheless, the current state of the art lacks a device that can isolate a desired number or range of biological particles from a liquid biological sample for optical visualization. Such devices are very useful in a point-of-care diagnostic environment by enabling rapid and low cost diagnostics.

  One potential application of microfluidic devices is to use in combination with new and reliable biomarkers for the diagnosis and treatment of conditions and diseases. For example, recent studies have shown that specific functional declines specific to neurological conditions such as Alzheimer's disease can be detected in peripheral cells including platelets. Recent results also indicate that these same changes are often an early precursor of the most aggravated AD disease, most often mild memory loss (“single symptomatic progressive amnesia”). It has also been shown to be detectable in platelets from subjects diagnosed with certain mild cognitive impairment (MCI).

  Because microfilters are compatible with current microfabrication techniques, they are well suited for the preparation of microfluidic samples and filtration can be accomplished by pressurization, capillary action, or other induced flow. Furthermore, the precise dimensional and geometric controls afforded by the micromachine allow the development of optimal filter designs that are not possible with conventional membrane filtration. For example, hollow fiber membranes (usually used for plasma separation) have a large flow channel diameter of 200 to 400 μm, but microfluidic channels are easily constructed with dimensions commensurate with blood cells. (Red blood cells are typically 6-7 μm in diameter and 1-2 μm for platelets). Microfilter devices can also be fabricated with precise pore dimensions and geometry; in contrast, the pore size and geometry of microporous membranes is often non-uniform. Finally, microfilter devices made of optically transparent materials may allow direct visualization of target pathologies or their labeling.

Various embodiments include a microfluidic device that includes an upper plate that includes an inlet in fluid communication with an optical measurement chamber in conjunction with a lower plate that includes one or more filter channels, wherein The surface of the adjacent optical measurement chamber is in fluid communication with one or more filter channels. In another embodiment, the top plate includes more than one inlet. In another embodiment, the top plate includes more than one optical measurement chamber. In another embodiment, the one or more filter channels are arranged in rows of parallel lines that are inclined in opposite directions in any two adjacent rows. In another embodiment, the one or more filter channels are less than 1.0 μm wide. In another embodiment, the lower plate is a capillary force.
In conjunction with a capillary eluent chamber that enhances the filter flow through the force. In another embodiment, the flow channel connects the inlet to the optical measurement chamber. In another embodiment, the lower plate is made of glass. In another embodiment, the top plate is made of glass. In another embodiment, one or more temperature control devices are adapted to adjust the temperature in the optical measurement chamber. In another embodiment, the optical measurement chamber is configured to retain molecules based on affinity, size, charge or mobility. In another embodiment, the optical measurement chamber is configured to hold within 5% of the target number of biological particles. In another embodiment, the optical measurement chamber is configured to hold within 2% of the target number of biological particles. In another embodiment, the optical measurement chamber is configured to hold within 1% of the target number of biological particles. In another embodiment, the device is adapted to regulate fluid flow from the inlet to the optical measurement chamber by pressure, valves, or other similar components.

  Other embodiments include a microfluidic kit for diagnosis of Alzheimer's disease and / or mild cognitive impairment, including a microfluidic diagnostic device; and instructions for use.

  Other embodiments include kits for the diagnosis of neurodegenerative diseases, including centrifugal diagnostic devices including optical microcentrifuge tube inserts and / or cuvettes; and instructions for use. In another embodiment, the neurodegenerative disease is Alzheimer's disease and / or mild cognitive dysfunction.

  Various embodiments obtain a sample from a subject; quantify the amount or activity of a target moiety in the sample by transferring the sample to a device as described in claim 1 herein; And a method of diagnosing a neurodegenerative disease, comprising comparing the amount or activity of the target moiety in the sample with the amount or activity of the target moiety in the standard, wherein the method compares the standard moiety with the standard A change in the amount or activity of the target moiety indicates that the subject has or develops a neurodegenerative disease. In another embodiment, the sample comprises an untreated sample. In another embodiment, the sample comprises a peripheral tissue sample. In another embodiment, the targeting moiety comprises a mitochondrial protein. In another embodiment, the sample does not contain mitochondrial isolates and / or selections. In another embodiment, the sample has not been subjected to high energy destruction techniques. In another embodiment, the sample has not been subjected to sonication, nitrogen cavitation, and / or lysis procedures. In another embodiment, quantifying the amount or activity of the target moiety comprises determining cytochrome oxidase activity in the tissue sample. In another embodiment, quantifying the amount or activity of the target moiety comprises determining the amount of amyloid precursor protein present in the untreated sample. In another embodiment, the sample comprises plasma. In another embodiment, the sample comprises platelet rich plasma (PRP). In another embodiment, the subject is a mammal. In another embodiment, the subject is a human. In another embodiment, the neurodegenerative disease is alcoholism, Alexander disease, Alpers disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), telangiectasia ataxia, Batten disease, bovine spongiform encephalopathy. (BSE), Canavan disease, cerebral palsy, Cocaine syndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, frontotemporal lobar degeneration, Huntington's disease, HIV-related dementia, Kennedy disease, Krabbe disease , Lewy body dementia, neuroborreliosis, Machado-Joseph disease, multiple system atrophy, multiple sclerosis, narcolepsy, Niemann-Pick disease, PD, Pelizaeus-Merzbacher disease, Pick disease, primary lateral sclerosis, Prion disease, progressive supranuclear palsy, refsum disease , Sandhoff disease, Schilder disease, subacute association degeneration secondary to pernicious anemia, Spielmeier-Forked-Sjogren-Batten disease, spinocerebellar ataxia, spinal muscular atrophy, Steele-Richardson-Olszewsky disease, And at least one of spinal cord fistulas. In another embodiment, the targeting moiety comprises a mitochondrial translocase subunit. In another embodiment, the targeting moiety comprises an amyloid precursor protein, or the fragment, complexed with a mitochondrial protein.

Other embodiments include obtaining a sample from a subject; quantifying the amount or activity of the target moiety in the sample by transferring the sample to a centrifuge device; and determining the amount or activity of the target moiety in the sample. A method of diagnosing a neurodegenerative disease, comprising comparing to the amount or activity of a target moiety in a standard, wherein a change in the amount or activity of the target moiety relative to the standard is Has a neurodegenerative disease or develops a neurodegenerative disease. In another embodiment, the centrifuge device includes an optical microcentrifuge tube insert and / or a cuvette.
For example, the present invention provides the following items.
(Item 1)
A microfluidic device, the microfluidic device comprising:
(A) an upper plate including an inlet in fluid communication with the optical measurement chamber;
The top plate comprises:
(B) a lower plate containing one or more filter channels;
Where,
(C) a microfluidic device, wherein a surface of the optical measurement chamber adjacent to the lower plate is in fluid communication with the one or more filter channels.
(Item 2)
The device of claim 1, wherein the top plate includes more than one inlet.
(Item 3)
The device of item 1, wherein the top plate comprises more than one optical measurement chamber.
(Item 4)
Item 2. The device of item 1, wherein the one or more filter channels are arranged in rows of parallel lines that are inclined in opposite directions in any two adjacent rows.
(Item 5)
The device of item 1, wherein the one or more filter channels are less than 1.0 μm wide.
(Item 6)
Item 2. The device of item 1, wherein the lower plate is associated with a capillary eluent chamber that enhances the flow of the filter via capillary forces.
(Item 7)
Item 2. The device of item 1, wherein a flow channel connects the inlet to the optical measurement chamber.
(Item 8)
Item 2. The device according to Item 1, wherein the lower plate is made of glass.
(Item 9)
Item 2. The device of item 1, wherein the upper plate is made of glass.
(Item 10)
Item 2. The device of item 1, wherein one or more temperature controllers are adapted to adjust the temperature in the optical measurement chamber.
(Item 11)
Item 2. The device of item 1, wherein the optical measurement chamber is configured to retain molecules based on affinity, size, charge or mobility.
(Item 12)
The device of claim 1, wherein the optical measurement chamber is configured to hold within 5% of the target number of biological particles.
(Item 13)
Item 2. The device of item 1, wherein the optical measurement chamber is configured to hold within 2% of the target number of biological particles.
(Item 14)
The device of claim 1, wherein the optical measurement chamber is configured to hold within 1% of the target number of biological particles.
(Item 15)
The device of claim 1, wherein the device is adapted to regulate fluid flow from the inlet to the optical measurement chamber by pressure, valves, or other similar components.
(Item 16)
A microfluidic kit for the diagnosis of Alzheimer's disease and / or mild cognitive impairment, comprising (a) a microfluidic diagnostic device; and (b) instructions for use.
(Item 17)
A kit for diagnosis of neurodegenerative disease, comprising (a) a centrifugal diagnostic device comprising an optical microcentrifuge tube insert and / or a cuvette; and (b) instructions for use.
(Item 18)
Item 18. The kit according to Item 17, wherein the neurodegenerative disease is Alzheimer's disease and / or mild cognitive impairment.
(Item 19)
A method of diagnosing a neurodegenerative disease comprising:
Obtaining a sample from a subject;
Quantifying the amount or activity of the target moiety in the sample by transferring the sample to the device of item 1; and determining the amount or activity of the target moiety in the sample from the target moiety in the standard A change in the amount or activity of the target moiety compared to the standard, wherein the subject has a neurodegenerative disease or develops a neurodegenerative disease Showing the way.
(Item 20)
20. A method according to item 19, wherein the sample comprises an untreated sample.
(Item 21)
20. A method according to item 19, wherein the sample comprises a peripheral tissue sample.
(Item 22)
20. A method according to item 19, wherein the targeting moiety comprises a mitochondrial protein.
(Item 23)
20. A method according to item 19, wherein the sample does not contain mitochondrial isolates and / or selections.
(Item 24)
20. A method according to item 19, wherein the sample has not been exposed to a high energy destruction technique.
(Item 25)
20. The method of item 19, wherein the sample has not been subjected to sonication, nitrogen cavitation, and / or lysis procedures.
(Item 26)
20. The method of item 19, wherein quantifying the amount or activity of the target moiety comprises determining cytochrome oxidase activity in the tissue sample.
(Item 27)
20. The method of item 19, wherein quantifying the amount or activity of the target moiety comprises determining the amount of amyloid precursor protein present in the untreated sample.
(Item 28)
20. A method according to item 19, wherein the sample comprises plasma.
(Item 29)
20. The method of item 19, wherein the sample comprises platelet rich plasma (PRP).
(Item 30)
20. A method according to item 19, wherein the subject is a mammal.
(Item 31)
Item 20. The method according to Item 19, wherein the subject is a human.
(Item 32)
The neurodegenerative diseases include alcoholism, Alexander disease, Alpers disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), capillary dilatation ataxia, Batten disease, bovine spongiform encephalopathy (BSE), Canavan disease , Cerebral palsy, cocaine syndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, frontotemporal lobar degeneration, Huntington's disease, HIV-related dementia, Kennedy disease, Krabbe disease, Lewy body dementia, neuroborreliosis, Machado-Joseph disease, multiple system atrophy, multiple sclerosis, narcolepsy, Niemann-Pick disease, PD, Pelizaeus-Merzbacher disease, Pick disease, primary lateral sclerosis, prion disease, progressive supranuclear palsy, refsum , Sandhoff disease, Schilder's disease, subacute association degeneration secondary to pernicious anemia, Spielmeier-För Transfected - Sjogren - Batten disease, spinocerebellar ataxia, spinal muscular atrophy, Steele - Richardson - Olszewski disease, and at least one of a spinal cord wax The method of claim 19.
(Item 33)
20. A method according to item 19, wherein the targeting moiety comprises a mitochondrial translocase subunit.
(Item 34)
20. A method according to item 19, wherein the targeting moiety comprises an amyloid precursor protein, or a fragment thereof, complexed with a mitochondrial protein.
(Item 35)
A method of diagnosing a neurodegenerative disease comprising:
Obtaining a sample from a subject;
Quantifying the amount or activity of the target moiety in the sample by transferring the sample to a centrifuge device; and the amount or activity of the target moiety in the sample to determine the amount or activity of the target moiety in the standard A change in the amount or activity of the target moiety compared to the standard, wherein the subject indicates that the subject has or develops a neurodegenerative disease .
(Item 36)
36. The method of item 35, wherein the centrifuge device comprises an optical microcentrifuge tube insert and / or a cuvette.

  Other features and advantages of the present invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, illustrating by way of example various embodiments of the invention.

  Exemplary embodiments are illustrated in the reference drawings. The embodiments and figures disclosed herein are intended to be considered illustrative rather than limiting.

FIG. 1 shows a schematic diagram of a microfluidic device that allows concentration measurement of a stained suspension cell sample according to embodiments herein. A common design may be a microfabrication technique, including a sandwich design where the features engraved on the surface become internal features when assembled. The device can include two chopped glass plates that, when assembled and bonded, form an internal network of interconnected channels and chambers. (A) discloses the upper plate in both a top view 100 and a side view 101. The upper plate includes an inlet 102 for injecting a sample suspended in buffer, and a flow channel 103 leading to an optical measurement chamber 104 above a filter channel 107 engraved in the lower plate. (B) discloses the lower plate in both a top view 105 and a side view 106. In one embodiment, 6 to 8 of these devices can be aligned on a chip designed in the form of a 96-well plate for reading with a standard microplate reader. FIG. 2 shows a schematic diagram of a microfluidic device that allows concentration measurements of stained suspension cell samples according to embodiments herein. A common design may be a microfabrication technique, including a sandwich design where the features engraved on the surface become internal features when assembled. (A) discloses the upper plate in both a top view 100 and a side view 101. The top plate includes an inlet 102 that is pierced (eg, a needle with a 21 G = 0.8 mm outer diameter). The top plate also includes a flow channel 103, 1 mm wide by 500 μm high, that leads to an optical measurement chamber 104 that covers a variable length transverse flow filter channel 107. (B) discloses the lower filter plate in both a top view 105 and a side view 106, the lower filter plate being dimensionally smaller than the upper plate. The filter channel 107 of the lower filter plate is a fishbone shape of a shallow cross-flow filter channel with a width of less than 1.0 μm and an indefinite depth. In one embodiment, there is also a lower capping plate that is featureless and dimensionally identical to the upper plate, including the capillary eluent chamber 108 formed by the lower capping plate or wicking absorbent. obtain. FIG. 3 shows an insert design that allows concentration measurements of stained suspension cell samples using microcentrifugation according to embodiments herein. The insert can act similarly to a microcentrifuge tube insert, where stained particles such as platelets can be reliably pelleted into the optical pocket of the insert for standardized optical measurements. . The above figure shows an optical measurement for an insert that may have a tube 110 with typical dimensions of a 1.5 mL tube, for example an inner dimension of 2 mm wide × 14 mm, a diameter of 0.5-1.0 mm The chamber 104 is shown. The figure also shows a support fin 109 that may be, for example, about 0.85 mm thick. In one embodiment described in further detail herein, the staining intensity of platelets is reduced in Alzheimer's disease patients, and in precursors, and this reduction can be reliably measured. FIG. 4 shows an insert cuvette 111 design as an accessory to enable insertion reading in a standard spectrophotometer, according to embodiments herein. In one embodiment, the cuvette may include a cuvette optical window 112. According to another embodiment, the design may also include a mounting slot 113 for capturing and directing the insert support fins 109. In another embodiment, the accessory may allow reading in a standard microplate reader. FIG. 5 shows a flowchart of an overall method for quantifying a target portion from a sample, according to embodiments herein. In one embodiment, the sample can be obtained from the subject 200, followed by staining the sample 201 and then transferring to a microfluidic device 202 where the carrier solution is retained while the stained sample is retained. 203, and then finally the optical density is measured and compared 204 with the standard. In another embodiment, the sample can be obtained from a subject 200, followed by staining the sample 201, then transferring to a centrifuge device 205, centrifuging to induce sedimentation of the stained sample 206, and finally optical density. And compare the density with the standard 207.

  All references cited herein are incorporated by reference in their entirety as if fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology, 3rd edition, J. MoI. Wiley & Sons (New York, NY2001); March, Advanced Organic Chemistry Reactions, Machinery and Structure 5th Edition, J. MoI. Wiley & Sons (New York, NY2001); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual Third Edition, Cold Spring Harbor Laboratory Press (Cold Spring Provides general guidance on.

  Those skilled in the art will recognize and be able to use many methods and materials similar or equivalent to those described herein in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

  As used herein, the term “diagnose” or “diagnosis” refers to determining the nature or identity of a condition or disease. Diagnosis can also be accompanied by a determination regarding the severity of the disease.

  As used herein, “prognosis” or “prognosis” refers to predicting the outcome of a disease.

  As used herein, “AD” means Alzheimer's disease.

  As used herein, “MCI” means mild cognitive impairment.

  As used herein, the term “PD” means Parkinson's disease.

  As used herein, “fluid connection” means a communication that includes a liquid communication such as water, blood, plasma, and the like.

  As used herein, “untreated sample” refers to a sample that has not been subjected to high energy disruption techniques, such as sonication or nitrogen cavitation. By way of example, the term is by no means so limited, but an untreated sample containing platelet-rich plasma can be subjected to filtration, purification, and / or isolation of the mitochondria itself prior to analysis for mitochondrial enzyme dysfunction. Absent.

  As used herein, the term “fishbone” pattern refers to a pattern of parallel lines that are inclined in opposite directions in any two adjacent rows.

  As used herein, the top and bottom plates of a microfluidic device can also refer to different sides or regions of a single combined device. For example, but not in any way limiting, a microfluidic device having upper and lower plates can refer to a single combined device having one face containing the inlet and the opposite face containing the various channels.

  As used herein, “microfluidics” refers to devices that allow the flow of fluids, suspensions, or suspended particles at various size and size scales.

Microfluidics and Flow Devices As disclosed herein, various embodiments within the present invention allow the inventors to connect the optical measurement chamber 104 and the fluidic fluid when the components are assembled. A microfluidic device having a filter channel 107 positioned to communicate with was fabricated. The filter channel 107 may allow fluid flow from the optical measurement chamber 104 while blocking the flow of suspended blood cells, for example. In another embodiment, the microfluidic device is described in FIGS. 1, 2, 3, and / or 4 herein.

  Embodiments of the invention include a microfluidic diagnostic device. In certain embodiments, the device can isolate biological particles from a liquid sample. In certain embodiments, the particles can be, for example, red blood cells, white blood cells, platelets, fibroblasts, cultured cells, and the like. Similarly, in certain embodiments, the liquid sample can be whole blood, whole blood fraction, plasma, urine, lymph, broth, medium, and the like.

  Embodiments of the present invention can be made from any suitable material, such as plastic, glass, silica, composites, and the like.

  In an embodiment comprising a plastic material, the plastic material may be, for example, acrylonitrile butadiene styrene plastic (ABS), acetal, acrylic (Perspex), acrylonitrile (nylon), cellulosic, fluororesin, high density polyethylene ( HDPE), low density polyethylene (LDPE), noryl, polyarylate, polyarylsulfone, polybutylene, polybutylene terephthalate (PBT), polycarbonate, polyester, polyetherimide, polyetherketone, polyethylene (polyten), polypropylene, polyallomer, polyethylene Terephthalate, polyimide, polyamide-imide, polyvinyl acetate (PVA), polyvinyl chloride (PVC), poly Styrene, polysulfone, styrene, such as ABS PTFE (Teflon), may comprise a thermoplastic material.

  In an embodiment comprising a plastic material, the plastic material is a thermosetting material such as, for example, alkyd polyester, allyl, bakelite, epoxy, melamine, phenols, polybutadiene, polyester, polyurethane, silicone, urea, etc. obtain. Similarly, the plastic material may include bioplastic. Bioplastics are not conventional plastics, often derived from petroleum, but are in the form of plastics derived from renewable biomass sources, such as vegetable oil, corn starch, pea starch, or microflora. Suitable bioplastic molds for use in embodiments of the present invention include, for example, polylactic acid (PLA) plastic, poly-3-hydroxybutyrate (PHB), polyamide 11 (PA11), bio-derived polyethylene, and the like.

  In certain embodiments, the device may consist of a single component, for example. In certain embodiments, the device may consist of a plurality of components, such as an upper plate and a lower plate. In some embodiments, the plurality of components may be made from the same or different materials. In certain embodiments, the components can be uniform or non-uniform and can include, for example, channels, tubes, chambers, spaces, mouths, and the like. In non-uniform embodiments, the channels, tubes, chambers, spaces, mouths, etc. have, for example, a depth or diameter of 1 μm, or a depth or diameter of 2 μm, or a depth or diameter of 4 μm, or a depth or diameter of It can be a consistent size, such as 10 μm or more. Similarly, in non-uniform embodiments, the channels, tubes, chambers, spaces, mouths, etc. are, for example, between 1 and 3 μm in depth or diameter, between 3 and 6 μm in depth or diameter, or depth or diameter. Between or above 6 and 10 μm, or depth or diameter between 1 and 2 mm, or depth or diameter between 2 and 5 mm, or depth or diameter between 5 and 10 mm, or It can be a range of sizes, such as exceeding.

  In some embodiments of multiple components, the multiple components may be sealed to form a single unit. In certain embodiments, the chamber of the device may capture a predetermined number, amount, or volume of target biological particles. Similarly, certain embodiments of the device may maintain capture of a predetermined number or amount of target biological particles. Similarly, in certain embodiments, the chamber of the device comprises 0.1%, 0.5%, 1%, 2%, 4%, 7%, 10%, of a predetermined number or amount of particles. Within 15%, or 20%, or any other desired amount may be captured.

  In certain embodiments, the predetermined number or amount is, for example, from 0 to 100 particles, or from 100 to 1000 particles, or from 1000 to 10,000 particles, or from 10,000 to 100,000 particles, or from 100,000. It can be in a range such as 1,000,000 particles, or 1,000,000 to 5,000,000 particles, or within any of these ranges, or beyond.

  In some embodiments, the optical measurement chamber 104 can be optically transparent so that biological particles within the chamber can be visualized. In certain embodiments, the visualization may be accomplished by any suitable method, such as a densitometric / spectrophotometric reader, a luminescence / fluorescence reader, etc. In certain embodiments, the size and arrangement of the measurement chamber can be optimized for a particular target biological particle.

  Embodiments of the present invention may include at least one filter channel 107 arranged to drain fluid from the optical measurement chamber 104.

In certain embodiments, the present invention provides a device comprising:
(A) a top plate including an inlet 102 in fluid communication with the optical measurement chamber 104; and;
(B) a lower plate comprising at least one filter channel 107;
(C) where the surface of the optical measurement chamber 104 adjacent to the lower plate is in fluid communication with at least one filter channel 107.

  In certain embodiments, the present invention provides a method of diagnosing the presence, absence, or severity of a disease, for example. Similarly, in some embodiments, the present invention provides a method of diagnosing the likelihood of developing or not developing a disease. In certain embodiments, the disease can be, for example, AD, PD, other neurodegenerative diseases, and the like. In certain embodiments, the method includes providing a biological sample, manipulating the sample so that certain characteristics of the sample can be visualized, subjecting the sample to a device of the invention, and Visualizing the sample may include.

  The advantages of that design include the use of small blood samples that are easily achievable, which can be processed and measured in less than 2 hours. The new microfluidic measurement device also enables rapid concentration (or fluorescence or luminescence) measurement of labeled samples. The reaction product in the device can be measured with a microplate reader after a simple reaction step-in a basic clinical laboratory, with the basic device, and possible use by minimally trained laboratory personnel. Designed for. Various embodiments can measure cellular functions that have been repeatedly found to be deficient in Alzheimer's disease brain, such as platelets and other tissues, and whole, unlysed cells—in other words, untreated Can be measured with high sensitivity without resorting to mitochondrial isolation, which is the biggest disruption in conventional ETC enzyme research. The devices are particularly useful for a wide range of dyes, reactions, and probes, including antibody-based technologies, and are modified for different cell types (eg, lymphocytes, culture lines) in clinical and research applications. Because it has, it has wide application in other diseases.

Diagnostic methods using mitochondria As disclosed herein, recent studies have shown that mitochondrial electron transport system (ETC) enzymes are functionally deficient in AD and mild cognitive impairment (MCI) showed that. These specific defects (especially in the enzyme cytochrome oxidase) were found in the brain and platelets of AD patients and other peripheral tissues (which are somewhat unacceptable; for example muscle). We have found a significant decrease in the function of cytochrome oxidase (C.O.) in platelet mitochondria isolated from subjects with MCI, which interferes with mitochondrial function and is a biomarker for disease. We support the view that it is an early and detectable systemic event in the pathophysiology of AD that can be used as:

In certain embodiments, the present invention provides methods for diagnosing neurodegenerative diseases, including:
(A) providing a tissue sample from a subject;
(B) quantifying the amount or activity of the target moiety in the cells of the sample; and (c) comparing the amount or activity of the target moiety in the cells of the sample with the amount or activity of the target moiety in the standard, Here, compared to the standard, a change in the amount or activity of the target moiety in the cells of the sample indicates that the subject has a neurodegenerative disease.

  In certain embodiments, the neurodegenerative disease is, for example, alcoholism, Alexander disease, Alpers disease, AD, ALS, telangiectasia ataxia, Batten disease (also known as Spielmeier-Forked-Sjogren-Batten disease). ), Bovine spongiform encephalopathy (BSE), Canavan disease, cerebral palsy, cocaine syndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, frontotemporal lobar degeneration, HD, HIV-related dementia, Kennedy disease, Krabbe disease , Lewy body dementia, neuroborreliosis, Machado-Joseph disease (spinal cerebellar ataxia type 3), multiple system atrophy, multiple sclerosis, narcolepsy, Niemann-Pick disease, PD, Pelizaeus Merzbacher disease, Pick disease, Primary lateral sclerosis, prion disease, progressive supranuclear palsy, refsum disease, sand Subacute associative degeneration secondary to Fuh disease, Schilder's disease, pernicious anemia, Spielmeier-Forked-Sjogren-Batten disease (also known as Batten disease), spinocerebellar ataxia (multiple forms with various characteristics) , Spinal muscular atrophy, Steele-Richardson-Olszewsky disease, spinal cord fistula.

  In some embodiments, the tissue sample can include, for example, amniotic fluid, blood, cerebrospinal fluid, plasma, serum, synovial fluid, other biological fluids, and the like. Certain embodiments may utilize fractionated samples, such as blood fraction, plasma fraction, and the like. Some embodiments may utilize platelet rich plasma (PRP). In embodiments utilizing fractionated samples, the fractionation can be accomplished using, for example, fractional centrifugation. In certain embodiments, the tissue sample can be obtained from a subject, for example, by amniocentesis, blood collection, spinal tap, and the like.

  In some embodiments, the tissue sample may include, for example, muscle cells, muscle fibers, or muscle tissue such as cardiac muscle tissue, smooth muscle tissue, or skeletal muscle tissue. In certain embodiments, the tissue sample can be obtained, for example, by biopsy.

  In certain embodiments, the subject can be a mammal such as a marsupial, a single hole, a placental mammal, and the like. In an embodiment utilizing a placental mammal, the placental mammal can be, for example, a human. In some embodiments, quantifying the amount or activity of the target moiety may be accomplished, for example, by histochemical techniques, immunochemical techniques, combinations thereof, and the like.

  In certain embodiments, quantifying the amount or activity of the target moiety can be, for example, amyloid beta (Aβ), amyloid precursor protein (APP), mitochondrial translocase (subunit TOMM40), APP-TOM40 complex. Can mean quantifying the presence of a protein, such as In some embodiments, quantification may be achieved by the use of antibodies reactive with the target moiety.

  In certain embodiments, quantifying the amount or activity of the target moiety may mean, for example, assessing enzyme activity. In some embodiments, this may include, for example, densitometry / spectrophotometric evaluation, fluorometric evaluation, histochemical labeling, and the like.

  In certain embodiments, the standard amount or activity of the target moiety can be determined from, for example, patient samples, literature, computer databases, or combinations thereof. In some embodiments, a change in the amount or activity of the target moiety can be, for example, an increase or decrease.

  In certain embodiments, the cell to be tested for the amount or activity of the target moiety can be a cell containing any mitochondria, such as, for example, a platelet.

  Certain embodiments of the present invention utilize a diagnostic test format, such as cuvettes, multiwell plates, microfilter devices, and the like. In some embodiments, the multi-well plate is, for example, 4 holes, 6 holes, 12 holes, 24 holes, 48 holes, 96 holes, 192 holes, 384 holes, 768 holes, 1536 holes, or more. Can be included. In some embodiments, the microfilter device may include, for example, a fill port, optionally a transparent measurement chamber, and at least one drainage channel. In some embodiments, the microfilter device may be made of plastic, glass, silica, and the like.

Kits Embodiments of the present invention are also kits for both the presence, absence, severity of neurodegenerative disease, diagnosis of the onset or potential for onset, and the preparation and use of microfluidic and centrifuge devices. Directed to. The kit is a collection of materials or components suitable for carrying out the method of the present invention. Accordingly, in some embodiments, the kits illustrate the methods of the invention as described in the examples above or below.

  The exact nature of the components comprised in the kit of the invention will depend on its intended purpose. For example, some embodiments are configured for the purpose of diagnosing a neurodegenerative disease. In one embodiment, the kit is configured specifically for the purpose of diagnosing AD. In another embodiment, the kit is configured specifically for the purpose of diagnosing PD. Alternatively, in some embodiments, the kit includes a device comprising an upper plate and a lower plate having an inlet 102 in fluid communication with the optical measurement chamber 104, as described above.

  Instructions for use can be included in the kit. “Instructions for use” typically describes techniques employed in using the components of the kit to obtain a desired result, such as diagnosing the presence of AD in humans. , Including specific expressions. Where necessary, the kit may also include diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, dressing materials or other as readily recognized by one skilled in the art. Including other useful components, such as useful equipment.

  The materials and components collected in the kit can be stored and provided to the practitioner in any convenient and suitable manner that maintains its operability and utility. For example, the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room temperature, refrigerated temperature, or frozen temperature. Such components are typically contained in suitable packaging material (s). As employed herein, the phrase “packaging material” refers to one or more physical structures used to contain the contents of a kit, such as an inventive device. The packaging material is constructed by well-known methods, preferably to provide a sterile, contaminant-free environment. The packaging material employed in the kit is customarily utilized in diagnostic assays. As used herein, the term “packaging” refers to a suitable solid matrix or material, such as glass, plastic, paper, foil, etc., that can hold individual kit components. Thus, for example, a package can be a plastic container used to contain an appropriate amount of an inventive device. The packaging material generally has an external label that indicates the contents and / or purpose of the kit and / or the components.

Diagnostic Protocol As disclosed herein, various embodiments include methods of diagnosing a subject's disease and / or condition by a combination of one or more of the following steps: (1) Subject Obtain a sample from 200; (2) Stain the sample 201; (3) Transfer to a microfluidic device 202, where (4) The carrier solution can pass 203 while the stained sample is held 203; And then (5) diagnosing the subject 204 by measuring the optical density and comparing the density to a standard.

  In one embodiment, sample staining 201 includes collecting blood from a subject in a centrifuge tube containing an anticoagulant. In another embodiment, the PRP is acidified by centrifuging the blood, followed by aspiration of the PRP (platelet rich plasma) layer, first adding ACD, and then adding ACD dropwise while checking the pH of the PRP. Turn into. In another embodiment, the PRP is diluted with phosphate buffered saline (PBS), centrifuged, and then the supernatant is transferred. In another embodiment, the supernatant is centrifuged to pellet the platelets and then resuspended in modified Tyrode's buffer. In another embodiment, platelets in modified Tyrode's buffer are added to a tube containing an equal volume of 2X reaction staining solution. In another embodiment, the tube is incubated, centrifuged, and the resulting pellet is resuspended and then transferred to a microfluidic device and / or centrifuge device for quantification.

  In one embodiment, transfer of the stained sample to the microfluidic device inlet 102 for quantification may be by manual injection, such as by syringe. In another embodiment, the movement may be in an automated manner, such as with an infusion pump or other device. In another embodiment, the stained platelet suspension flows through the internal channel (s) and into the optical measurement chamber 104 above the filtration channel 107 under the pressure of the application (eg, syringe or pump). . As described herein, the filtration channels 107 can be aligned to maximize the passage of platelet carrier solution (ie, buffer) while retaining the platelets in the optical measurement chamber 104. In another embodiment, the filtration channel 107 is designed to enhance the passage of the carrier solution by capillary force and thereby enhance the platelet packing density in the optical measurement chamber 104. Thus, in another embodiment, draining of the carrier solution and concomitant platelet filling can be achieved by two forces: the force of application and the capillary force in the drainage channel. As the carrier solution is withdrawn from the optical measurement chamber 104, the platelet packing density reaches a maximum and therefore constant value appropriate for control of subsequent staining density measurements in the platelets.

  In another embodiment, the optical density of the optical measurement chamber 104 can be measured at an established wavelength and the results compared to tabulated values. In another embodiment, the optical density of the optical measurement chamber 104 may be measured in conjunction with the included standard reference material. This measurement can be performed, for example, on a manufactured plate or frame having the dimensions of a standard microplate (eg 96-well microplate) with the optical measurement chamber 104 aligned with the standard coordinates of a multiwell microplate. It can be accomplished by containing an Idic device or multiple devices for higher economy. Thus, in another embodiment, standard microplate readers can be used to select specific well coordinates and specific wavelengths as they are accustomed to and perform measurements. Another embodiment is a microfluidic device wherein the optical measurement chamber 104 is vertically and in a frame having the dimensions of a standard spectrophotometer cuvette so that it is aligned with the standard beam of a spectrophotometer. Can be incorporated. In designs targeted for measurements in a spectrophotometer or a microplate spectrophotometric reader, spectrophotometry at a specified wavelength is used as a surrogate for concentration measurement. Thus, in another embodiment, an alternative wavelength may be specified and if the specified wavelength cannot be used for use in a non-monochromator-equipped spectrophotometric device or otherwise May or may not incorporate various correction factors. Another embodiment may incorporate similar microfluidic design features, but allows direct measurement in an optical densitometer. Each of the described embodiments is equally effective and can be provided as a kit, each targeting a user's available device (eg, “microplate reader diagnostic kit”, “spectrophotometer diagnostic kit” ").

  As further disclosed herein, various embodiments include methods of diagnosing a subject's disease and / or condition by a combination of one or more of the following steps: (1) subjecting a sample 200 which can be obtained from the body; (2) subsequently staining the sample 201; and (3) transferring the stained sample to a centrifuge device 205; (4) centrifuging to induce sedimentation of the stained sample 206; and then (5 ) Measure the optical density and compare 207 its concentration with the standard.

  In another embodiment, the transfer of the stained sample to the centrifuge device can include transfer to a tube insert, as described in FIG. 3 herein. In another embodiment, the tube insert is placed in a standard 1.5 ml microcentrifuge tube 110, the stained platelet suspension is transferred to the tube insert (eg, by pipette), and the tube and insert Is centrifuged at a determined speed (eg 2000 × g) for a determined time (eg 15 minutes) to induce sedimentation of the stained platelets into the optical measurement chamber 104, or “optical pocket”. Thus, under a constant centrifugal force, the stained platelet packing density in the measurement chamber reaches a maximum and therefore a constant value appropriate for the control of subsequent measurement of the staining concentration in the platelets.

  In another embodiment, the absorbance of the optical measurement chamber is measured at an established wavelength (eg, 330 nm) and the results are compared to tabulated values or measured in conjunction with a standard reference material included. In another embodiment, the measurement chamber is aligned with the standard coordinates of a multi-well microplate to provide this measurement, for example, insertion of the tube insert into the frame, and a standard microplate (eg 96 wells). This can be achieved by providing a manufactured plate or frame having the dimensions of (microplate). Thus, in any standard microplate reader, users can select specific well coordinates and specific wavelengths as they are accustomed to and make measurements. Another embodiment may incorporate a frame having the dimensions of a standard spectrophotometer cuvette 111 so that its optical measurement chamber 104 is aligned with the standard beam of the spectrophotometer upon insertion. In designs targeted for measurements in spectrophotometers or microplate spectrophotometric readers, spectrophotometric measurements at specified wavelengths are used as surrogates for concentration measurements. Thus, in another embodiment, alternative wavelengths may be specified and various correction factors may be used for use in non-monochromator equipped spectrophotometric devices, or otherwise where the specified wavelength cannot be used. May or may not be embedded. Another embodiment may incorporate similar microfluidic design features, but allows direct measurement in an optical densitometer. Each of the described embodiments is equally effective and can be provided as a kit, each targeting a user's available device (eg, “microplate reader diagnostic kit”, “spectrophotometer diagnostics”). kit").

  Those skilled in the art will recognize and be able to use many methods and materials similar or equivalent to those described herein in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

  The following examples are provided to better illustrate the claimed invention and are not to be construed as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for illustrative purposes and is not intended to limit the invention. One skilled in the art can develop equivalent means or reactants without exerting the inventive ability and without departing from the scope of the present invention.

Example 1
Kit designs that provide simple and low cost histochemical evaluation Recent studies have shown that certain neurological declines (such as in Alzheimer's disease) can be detected in peripheral cells such as platelets. Necessary for activation and adhesion by histologically staining the activity of the enzyme of interest without activating the platelets (ie keeping the whole platelets in suspension during the procedure) Independent of complex methods, platelets can be forced into a tissue-like orientation for measuring the concentration of reaction products using microfluidic devices. This in turn eliminates the need for secondary control assays, and there is a reduction in the number of separate reagents and other required supplies, reducing overall cost and assay complexity. For example, we do not require a commercial separation tube with a separation medium (eg, Sigma-Aldrich Accuspin, Vacutainer CPT), but a general low cost tube (eg, an empty ACD tube; Using a differential centrifugation in (examined and confirmed by staining for) a pure platelet preparation can be achieved. We tested and eliminated the normal antiplatelet activator prostaglandin-1 and apyrase, relying solely on acid-citrate-dextrose anticoagulant.

  We designed a microfluidic device that captures / filters stained platelets in a uniform array in an optical measurement chamber that measures the stained product with the highest sensitivity while allowing the carrier buffer to pass through. did. The device provides unprecedented sensitivity in the measurement of stained products and thus unprecedented sensitivity and accuracy in the measurement of enzyme activity itself. The device is also of high quality, with or without simple modifications, for optical measurements in any number of ways (eg fluorescence, luminescence) across many types of suspension cells (eg lymphocytes, cancer cells) It can be used as a cell filter. Thus, in both clinics and laboratories, the number of possible applications of this type of device is almost infinite.

Example 2
Microfluidic plate design The general microfluidic plate design allows concentration measurements of stained suspension cell samples. Typical designs incorporate microfabrication techniques, including a sandwich design that forms features that are carved on the surface when assembled. The device can have, for example, two chopped glass plates that, when assembled and bonded, form an internal network of interconnected channels and chambers. The top plate can include an inlet for injection of the sample suspended in the buffer and a passage channel to the measurement chamber. The measurement chamber, or optical measurement chamber, is above the microfiltration channel carved in the lower plate. Six to eight of these devices can be arranged on a chip designed in the form of a 96-well plate, for example, for reading with a standard microplate reader.

Example 3
Design features of microfluidic plate designs The design is primarily a simple pass application, and the key test parameters are flow and filtration, ie the lack of “clogging” and the ability to produce a uniform measurement field. In platelet histochemistry studies, maintenance of anticoagulation successfully prevented platelet aggregation and activation during histochemical procedures. A further parameter is the filtration of the carrier buffer from the measurement chamber so that the platelets form a uniform layer that can be subjected to concentration measurements. This is important for one of the simplest variants of this design: in order to obtain a uniform array of platelets within the measurement chamber, the user relies solely on “platelet density” as an internal control 2 The use of subsequent control assays can be dispensed with.

  a. The inlet can be a mold commonly used in microfabricated designs.

  b. Flow channel. A flow channel allows unimpeded flow of the suspension into the measurement chamber. For example, modifications to the flow channel can be utilized, such as the incorporation of a microfabricated pressure control valve, that can prove useful for establishing consistency in platelet packing density.

  c. Measuring chamber. In one embodiment, the dimensions of the measurement chamber require manufacturing the device from a highly rigid material (eg, glass rather than plastic that is usually microfabricated) for two main reasons: ) Reliable platelet packing density cannot be achieved in flexible chambers, 2) The proposed dimensions (diameter: height / depth) can cause collapse of the chamber with soft materials. In one embodiment, the chamber diameter can be up to 3 mm. Measurement of reaction products in the visual spectrum requires optical clarity above and below the sample and can therefore use glass. An important factor in absorbance or densitometric evaluation is the scattering of light from an irregular surface (ie tissue); in the tissue staining application described herein, this is the Macroscale assessments (eg, brain area analysis) that are minimized by use and typically performed are relatively insensitive to any residual scatter. Lab, such as a standard but more sensitive microplate reader, rather than a new assembly of light sources, optics, and detectors that the end user must purchase Light scattering by an irregular sample surface becomes much more important when it is desired to measure with a device. In one embodiment, this may be addressed in one of two ways: 1) minimize the tissue thickness (chamber depth) that the beam must pass through, and 2) the inventor The glass surface of these devices is used to force the sample into almost perfect vertical and regular alignment with the beam.

  d. Microfiltration. The microfiltration channel device is designed to passively filter plasma from whole blood using only capillary action for downstream chemistry. The design allows for reliable filtration of platelets in suspension and captures them in the measurement chamber without any clogging uniformly in any given unit of application. Also, rather than relying solely on the front applied pressure (injection force) to facilitate cell filling, “pull” the platelet suspension into the chamber, and to maximize filling uniformity, It can also influence the posterior capillary force. Thus, rather than simply present as a simple grooved drain or filter, the bottom plate filtration channel plays an active role in preventing clogging and increasing cell density uniformity. obtain.

Example 4
Research to prove the applicability of the technology First, the maintenance of platelets in suspension was evaluated. The degree of anticoagulation required to maintain platelets without activation and binding was evaluated, and redundant application of platelet inhibitors such as apyrase and prostaglandin (PGE1) was stopped. This further reduces both the complexity and cost of the assay. Second, DAB reactivity with non-cytochrome c oxidase was evaluated. In most DAB labeling applications, peroxidase activity is the largest cause of background DAB reactivity, but we have used potassium cyanide inhibition of cytochrome c oxidase to elucidate other reaction mechanisms. Have shown that its activity is relatively low (and decreases by increasing platelet washing).

Example 5
Measurement of cytochrome C oxidase activity-staining of untreated sample 10 mL of whole blood is collected from a test subject into a centrifuge tube or tube containing an anticoagulant. The blood is centrifuged at 300 RCF for 15 minutes, and then the PRP (platelet rich plasma) layer is aspirated into the 15 mL tube (s).

The PRP is acidified to pH 6.5 by first adding 300 μL of ACD and then adding ACD dropwise while checking the pH of the PRP. Next, the PRP is diluted to 5 mL with phosphate buffered saline (PBS). The PRP is then centrifuged at 600 RCF for 10 minutes at room temperature. The supernatant is then transferred to a new 15 mL tube and the pellet is discarded.

  The supernatant is centrifuged at 2000 RCF for 10 minutes to pellet the platelets, which are then resuspended in 300 μL of modified Tyrode's buffer prepared as follows per 8 ml blood collection:

A staining solution (2x) is prepared as follows:

The reaction staining solution is then heated to 37 ° C.

  Platelet in Tyrode's solution is added to a tube containing an equal volume of double reaction staining solution. The tube is closed and incubated in a 37 ° C. water bath for 1 hour. The tube is then centrifuged at 1600 RCF for 10 minutes at room temperature.

  The resulting pellet can be resuspended and then transferred to either a microfluidic device or a centrifuge device for quantification.

Example 6
Measurement of cytochrome C oxidase activity-transfer to microfluidic device After following the protocol as outlined in Example 5 above, the resulting pellet was resuspended and the microparticles described herein were Transfer to fluidic device. The application of stained platelets can be obtained by manual infusion by means of a syringe or in an automated manner, such as by an infusion pump or other device. The stained platelet suspension flows under the pressure of the application (eg syringe or pump) through the internal channel (s) to the measurement chamber above the microfiltration channel. The microfiltration channels are aligned to maximize the passage of platelet carrier solution (ie buffer) while maintaining the platelets in the measurement chamber. The microfiltration channel is designed to enhance the passage of the carrier solution by capillary forces and thereby enhance the platelet packing density in the measurement chamber. Thus, drainage of the carrier solution and concomitant platelet filling is achieved by two forces: the force of application and the capillary force of the drainage channel. Thus, as the carrier solution is withdrawn from the measurement chamber, the platelet packing density reaches a maximum and therefore constant value suitable for control of subsequent measurement of staining density within the platelets.

  The optical density of the optical measurement chamber is then measured at an established wavelength (eg 330 nm) and the results compared to the tabulated values or measured in conjunction with the included standard reference material To do. This measurement can be performed, for example, by aligning the measurement chamber with the standard coordinates of a multi-well microplate, and by connecting a microfluidic device, or multiple devices for higher economy, to a standard microplate (eg, a 96-well microplate). This can be achieved by placing it in a manufactured plate or frame having the dimensions of Thus, in any standard microplate reader, users can select the specific well coordinates and specific wavelengths they are accustomed to and perform measurements. Another embodiment may incorporate a microfluidic device that is vertically and in a frame having the dimensions of a standard spectrophotometer cuvette so that the measurement chamber is aligned with the standard beam of the spectrophotometer. In designs targeted for measurements in spectrophotometers or microplate spectrophotometric readers, spectrophotometric measurements at specified wavelengths are used as surrogates for concentration measurements. Alternate wavelengths may be specified and various correction factors may or may not be incorporated for use in non-monochromator-equipped spectrophotometric devices or otherwise where the specified wavelengths are not available . Another embodiment may incorporate similar microfluidic design features, but allows direct measurement in an optical densitometer. Each of the described embodiments is equally effective and can be provided as a kit, each targeting a user's available device (eg, “microplate reader diagnostic kit”, “spectrophotometer diagnostics”). kit").

Example 7
Measurement of cytochrome C oxidase activity-transfer to a centrifuge device After following the protocol as outlined in Example 5 above, the resuspended pellet was inserted into the microcentrifuge tube insert described herein. Move to. Place the tube insert into a standard 1.5 ml microcentrifuge tube. The stained platelet suspension is transferred to the insert (eg, by pipette) and the tube is capped. The tube and insert are centrifuged at a determined speed (eg 2000 × g) for a determined time (eg 15 minutes) to induce sedimentation of the stained platelets into the measuring chamber, or “optical pocket”. Thus, under a constant centrifugal force, the stained platelet packing density in the measurement chamber reaches a maximum and therefore a constant value appropriate for the control of subsequent measurement of the staining concentration in the platelets.

  The optical density of the optical measurement chamber is then measured at an established wavelength (eg 330 nm) and the results compared to tabulated values or measured in conjunction with the included standard reference material. To provide this measurement, for example, to insert the tube insert into the frame, the measurement chambers are aligned with the standard coordinates of a multi-well microplate and the dimensions of a standard microplate (eg 96-well microplate) are measured. This can be achieved by providing a manufactured plate or frame having. Thus, in any standard microplate reader, users can select specific well coordinates and specific wavelengths as they are accustomed to and make measurements. Another embodiment is to dimension the standard spectrophotometer cuvette (as shown in FIG. 4 herein) so that the measurement chamber is aligned with the standard beam of the spectrophotometer upon insertion. You can incorporate the frame you have. In designs targeted for measurements in spectrophotometers or microplate spectrophotometric readers, spectrophotometric measurements at specified wavelengths are used as surrogates for concentration measurements. Alternate wavelengths may be specified and various correction factors may or may not be incorporated for use in non-monochromator-equipped spectrophotometric devices or otherwise where the specified wavelengths are not available . Another embodiment may incorporate similar microfluidic design features, but allows direct measurement in an optical densitometer. Each of the described embodiments is equally effective and can be provided as a kit, each targeting a user's available device (eg, “microplate reader diagnostic kit”, “spectrophotometer diagnostics”). kit").

Example 8
Assay Optimization The assay optimization described herein 1) increase the signal-to-noise ratio (reduce background, increase measurement sensitivity), 2) reduce cost (Eliminating duplication or unnecessary addition of chemicals), and 3) reducing time (increasing reaction rate).

  a. Linearity of reaction towards optimization of staining substrate and time. One critical difference between the application of DAB staining for enzyme localization and DAB staining for quantification of enzyme activity is the intensity that drives staining. For any reliable quantification, such as routine tissue histochemical assessment, the degree of DAB staining is maintained at a non-saturated level. In contrast, many researchers maximize DAB staining just to indicate the presence of an enzyme, similar to immunological techniques. The use of DAB as described herein allows it to be used as a continuous and quantitative marker, not just qualitative; the resulting concentration measurement results can be used for enzyme turnover (ie, Can be converted linearly to, for example, the moles of enzymatically reduced cytochrome c). Based on this assumption, the assay can be tested for response linearity over time after any significant modification to the staining parameters. Maximizing the signal without saturation can be sought; the slope of the optical density “plateaus” or decreases over time indicates saturation and reduces staining time. Cobalt chloride or other metals for enhancement are also tested as DAB sensitizers and incorporated into the protocol if they reduce staining time while maintaining / increasing linearity of optical density with signal and time obtain.

  b. Cell permeation treatment. Histochemical staining with DAB is enhanced by the use of DMSO, allowing for increased cell permeability while maintaining membrane integrity. It can be assessed whether DMSO is required and whether increasing DMSO during staining allows for shorter incubation times. After such changes, the linearity of the reaction can also be evaluated as described above.

  c. Enzyme specificity. This can be tested by specific cytochrome c oxidase inhibition by potassium cyanide (KCN). KCN is added simultaneously with DAB in the comparative assay and any remaining DAB reactivity is considered background, non-specific activity.

Example 9
Design choices and modifications a. Internal control assay for cell number or content. If a uniform cell packing density cannot be reliably achieved, a simultaneously applied secondary assay can also be used in combination with various embodiments of the device for the measurement of mitochondrial content or similar variables. . There are many choices. Since the combination of succinate dehydrogenase / cytochrome c oxidase staining is often used in the determination of mitochondrial disease in muscle fibers, histochemical staining of succinate dehydrogenase is a natural choice. Succinate dehydrogenase is typically stained with nitroblue tetrazolium which produces a blue stain, which can be distinguished spectrophotometrically from the brown diaminobenzidine reaction produced from cytochrome c oxidase. However, other options are alternative modes such as fluorescence or emission that do not interfere with the overall optical density of the preparation. For example, Mitotracker Green FM (Invitrogen) can be employed reliably in this application. It can be quickly applied to cells in suspension, where it generally stains platelets (possibly due to a dense platelet inner membrane system) and thus potentially provides a measure of cell density. It was found to be obtained. In other cell types, it is likely to fluoresce only in mitochondria and thus provide a reasonable measure of mitochondrial concentration. These are just two examples considered, but if the cell density is insufficient as a control, it can be expected that another control could be used instead in the appropriate course without any major delay.

  b. Internal control assay for staining intensity. Even if a single staining mode (eg DAB) can be relied upon, the variability of staining results due to operator error must still be considered in most situations. Inaccurate incubation temperatures are probably the most common error, and it does not necessarily cause an absolute failure of the assay, and thus rapid detection of inaccurate results. Extreme values in the range of possible optical density values can be easily ruled out as errors (ie very light staining = low temperature; very dark staining = high temperature), but milder errors are false negatives or False positives can occur. Thus, the true temperature sensitivity of the assay can be assessed (to which an incubator / water bath temperature record can be applied) to allow modification. In addition, it may include temperature sensitive artificial reactants (eg, peroxidase contained in a permeable matrix) that can be stained in parallel with the patient sample as an indicator of appropriate conditions or as an internal correction factor for assay results. However, this increases the complexity and cost of the assay.

Example 10
Histochemical measurement of cytochrome C oxidase activity using a 96-well plate 10 mL of whole blood is collected from a test subject into a centrifuge tube. The blood is centrifuged at 300 RCF for 15 minutes, and then the PRP (platelet rich plasma) layer is aspirated into the 15 mL tube (s).

PRP is acidified to pH 6.5 by first adding 300 μL of ACD and then adding ACD dropwise while checking the pH of the PRP. Next, the PRP is diluted to 5 mL with phosphate buffered saline (PBS). The PRP is then centrifuged at 500 RCF for 10 minutes at room temperature. The supernatant is then transferred to a new 15 mL tube and the pellet is discarded.

  The supernatant is centrifuged at 2000 RCF for 10 minutes to pellet the platelets, which are then resuspended in 300 μL of modified Tyrode's buffer prepared as follows per 8 ml blood collection:

A staining solution (2x) is prepared as follows:

The reactive dyeing solution is then heated to 37 ° C. Platelet in Tyrode's solution is added to a tube containing an equal volume of double reaction staining solution. Close the tube and incubate in a 37 ° C. water bath for 1 hour. The tube is then centrifuged at 1600 RCF for 10 minutes at room temperature.

  The resulting pellet is resuspended and 40 μl is transferred to a well of a 96-well plate round bottom microplate. The microplate is refrigerated and individual wells are allowed to settle overnight.

  The OD is then measured at the determined wavelength (eg 330 nm) and the result compared to the tabulated value.

Example 11
Histochemical measurement of cytochrome C oxidase activity using a microfilter device 8.5 mL whole blood is collected from a test subject into a centrifuge tube. The tube is centrifuged at 300 RCF for 15 minutes, and then the PRP (platelet rich plasma) layer is aspirated into a 15 mL tube.

PRP is acidified to pH 6.5 by first adding 300 μL of ACD and then adding ACD dropwise while checking the pH of the PRP. Next, the PRP is diluted to 5 mL with phosphate buffered saline (PBS). The PRP is then centrifuged at 600 RCF for 10 minutes at room temperature. The supernatant is then transferred to a new 15 mL tube and the pellet is discarded.

  The supernatant is centrifuged at 2000 RCF for 10 minutes to pellet the platelets, which are then resuspended in 300 μL of modified Tyrode's buffer prepared as follows per 8 ml blood collection:

A staining solution (2x) is prepared as follows:

The reactive dyeing solution is then heated to 37 ° C.

  Platelet in Tyrode's solution is added to a tube containing an equal volume of double reaction staining solution. Close the tube and incubate in a 37 ° C. water bath for 1 hour. The tube is then centrifuged at 1600 RCF for 10 minutes at room temperature.

  The resulting pellet is resuspended and 40 μl is transferred to a microfiltration device as previously described. Add 40 μl of the above to the filling port of the device.

  The OD is then measured at the determined wavelength (eg 330 nm) and the result compared to the tabulated value.

  Various embodiments of the invention are described in the detailed description above. While these descriptions directly describe the above embodiments, those skilled in the art will envision modifications and / or variations on the specific embodiments shown and described herein. Any such modifications or variations that fall within the scope of this description are intended to be included therein as well. Unless expressly stated, it is the intent of the inventors that the terms and phrases herein and in the claims are given their ordinary and customary meaning to those of ordinary skill in the applicable field (s). It is.

  The foregoing description of various embodiments of the invention known to the applicant at this time of filing this application is presented and is intended for purposes of illustration and description. This description is not intended to be exhaustive or to limit the invention to the precise form disclosed, and many modifications and variations are possible in light of the above teaching. The described embodiments are provided to illustrate the principles of the present invention and its practical application, and to those skilled in the art in various embodiments suitable for various embodiments and for the particular use contemplated. It is useful to make modifications to allow the invention to be utilized. Accordingly, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.

  While specific embodiments of the invention have been shown and described, it will be appreciated that changes and modifications may be made based on the teachings herein without departing from the invention and its broader aspects, and thus It will be apparent to those skilled in the art that the appended claims include all such variations and modifications within the true spirit and scope of the invention. Further, it is understood that the invention is solely defined by the appended claims. In general, terms used herein, and particularly in the appended claims (eg, the body of the appended claims), are generally intended as “open” terms (eg, The term “including” should be interpreted as “including but not limited to”, the term “having” should be interpreted as “having at least”, “including ( the term “includes”) should be interpreted as “including but not limited to”). If a specific number of details of a claim introduced is intended, such intention is clearly detailed in that claim, and in the absence of such detail, such intention is It is further understood by those skilled in the art that it does not exist. For example, as an aid to understanding, the following appended claims may include use of the introductory phrases “at least one” and “one or more” to introduce a detailed description of the claims. However, the use of such a phrase does not imply that the indefinite article “a” is used even if the same claim contains the indefinite article such as “1 or more” or “at least one” and “a” or “an”. Introducing a claim detail by "or" an "means limiting any particular claim that contains such introduced claim details to an invention that contains only one such detail. Should not be construed (eg, “a” and / or “an” should typically be construed to mean “at least one” or “one or more”); The same is true for the use of definite articles used to introduce predicates. In addition, even though details of a particular number of introduced claims are clearly detailed, those skilled in the art will interpret such details as typically meaning at least the number detailed. (E.g., a detailed description of "two details" without other modifiers typically means at least two details, or more than one detail) ).

  Accordingly, the invention is not limited except as by the appended claims.

Claims (1)

The invention described herein.